This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The lymphatic system originates from the blood vascular system. Lymphatic endothelial cells (LECs) differentiate from blood endothelial cells (BECs), and this lineage decision as well as the maintenance of the LEC phenotype are tightly controlled processes, which is essential for development and functions of the two discrete vascular compartments. The transcription factor Prox1 is a binary regulator that promotes LEC lineage identity and suppresses BEC phenotype. However, how it functions is not well understood. We observed that mice lacking podoplanin, which is highly expressed in LECs, exhibit blood-filled lymphatic vessels that closely resemble those seen in time-specific deletion of Prox1 (inducible Prox1[unreadable]/[unreadable]) mice. We hypothesize that podoplanin, like Prox1, is critical in regulating endothelial cell identity. To test this, we propose two aims. Aim 1 will determine the embryonic and postnatal stages at which podoplanin controls LEC identity using mice with time-specific podoplanin deficiency in endothelial cells. Aim 2 will determine whether forced expression of podoplanin suppresses BEC identity in vivo using a transgenic line that over-expresses podoplanin in BECs and LECs (TetO-PdpnEC). Increased expression of Prox1 and podoplanin is associated with malignant transformation of BEC-derived angiosarcoma with a mixed BEC and LEC identity. We will investigate whether over-expression of podoplanin in endothelial cell lines (EOMA and Py-4-1) induces formation of aggressive angiosarcoma in vivo.